The goal of the proposed research is to identify the functional groups at the active site of the mitochondrial F1-ATPase that participate in substrate binding and in the bond-making and bond-breaking steps that occur during catalysis. Analogues of ATP that are potential affinity labels for the enzyme will be synthesized for these studies. These analogues will also be used to determine if a nucleotide binding site exists in the enzyme in addition to the one that we have identified in the beta subunit with p-F-sulfonyl(14C)benzoyl-5'-adenosine. In addition studies on the topography of the ATP synthetase complex will be carried out. These studies will involve the modification of soluble coupling factors with soluble mercaptoalkylimidoesters followed by modification with Ellman's reagent to produce zwitterionic mixed disulfides that reduce the positive charge on the lysine residues to which they are attached by one. The zwitterionic derivatives will be fractionated by ion exchange chromatography and the resolved species will be converted to mixed disulfides which contain nitroaryl azides by reaction with 4-azido-2-nitrobenzenemercaptan. The modified coupling factors will be photolyzed in the presence of the F1-ATPase and depleted membrane preparations. Specific cross-links will be identified by two dimensional polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate in which the cross-links are cleaved after the first dimension. Since it contains the catalytic site for ATP synthesis and hydrolysis, the complete amino acid sequence of the beta subunit is being determined.